1. The Field
The present application relates to certain compounds and to methods for the preparation and the use of certain compounds in the fields of chemistry and medicine.
2. Description of the Related Art
Hepatitis C virus (HCV), a positive-strand RNA virus, is a leading cause of chronic liver disease, with over 170 million people infected worldwide. According to the Centers for Disease Control (CDC), chronic HCV infection currently affects more than 3 million Americans and causes 10,000 to 12,000 deaths a year in the United States. The CDC estimates that the annual mortality rate will increase to 38,000 by the year 2010, surpassing the number of deaths attributed annually to HIV/AIDS. HCV infection is also the leading indication for liver transplantation.
There is neither a vaccine nor a direct antiviral drug available to treat or prevent the spread of HCV. The current standard of care for chronic HCV consists of a combination of injected interferon-alpha and the nucleoside analogue ribavirin. The significant side effects associated with both drugs render it difficult to sustain therapy over prolonged periods of time. Many patients require additional drugs to treat adverse effects of interferon, further increasing the cost and the risk of additional side effects. As a result, poor compliance with the course of HCV therapy decreases the patient response rate. Importantly, current HCV therapies are directed at stimulating the immune system but do not directly target the virus. Consequently, sustained virus elimination is not achieved in more than half of the treated patients even after six months of therapy. Therefore, novel drugs are required to treat HCV infection by directly acting on viral targets.
A majority of drug discovery research for HCV has focused on the viral proteins which include structural and nonstructural (NS) targets. Among the latter, the NS3-4A protease and the NS5B RNA-dependent RNA polymerase are in the focus of many antiviral discovery programs, paralleling past efforts in the somewhat corresponding protease and reverse transcriptase targets of human immunodeficiency virus (HIV). As with HIV, the high genetic variability of HCV poses a significant challenge for the development of antiviral mono-therapies. The low fidelity of the HCV NS5B polymerase facilitates the emergence of viral variants, including six major genotypes and a large number of subtypes. Rapid selection of resistant virus populations is expected under mono-therapy treatment regimes. Thus, combination of several drugs to attack distinct viral targets will be mandatory for successful HCV therapy.
Since the HCV genome contains several highly conserved cis-acting RNA elements, the repertoire of protein targets for antiviral intervention may be expanded by RNA targets. Structured functional elements of the HCV genome that are candidate drug targets have been identified in the 5′ and 3′ nontranslated regions (NTR) and in the coding region of the NS5B polymerase.
The 5′ NTR stretches over 341 nt of which the first 40 are essential for RNA replication. The 330 nt region immediately flanking the reading frame for the viral genes contains an internal ribosome entry site (IRES) which mediates translation initiation of the viral message via a 5′ cap-independent mechanism. The IRES RNA binds directly to the host cell 40S ribosomal subunit and initiates protein translation in the absence of most initiation factors. Recruitment of the small ribosomal subunit to the HCV message is driven entirely through the high affinity of the IRES RNA-40S interaction. The IRES RNA sequence is one of the most conserved regions of the entire viral genome and adopts a highly ordered secondary structure.
Most of the IRES subdomains are critical for translation initiation, including the stem-loops, a helix between subdomains II and III, a proposed pseudoknot involving loop IIIf and the single-stranded regions that flank subdomain IIb, and a stem-loop containing the start AUG codon. The three-dimensional architecture of the IRES RNA is dominated by the independently-folding subdomains that adopt specific folds in the presence of physiological concentrations of metal ions. Since the single-stranded stretches between the subdomains are flexible, the IRES element becomes three-dimensionally ordered only after binding to the 40S ribosomal subunit. Three-dimensional structures of individual subdomains have been determined by crystallography and NMR, including the subdomains II and IIIa-e which revealed unique RNA architectures that might be exploited for small-molecule recognition. Based on its importance for viral replication and its high conservation the HCV IRES element has been discussed as a potential target for therapeutic intervention. For example, it has been observed that mutational stabilization of stem-loop IV, which contains the initiator AUG codon of the HCV polyprotein, prevented translation of the viral mRNA, suggesting an approach for the development of IRES RNA-stabilizing ligands as antivirals. Validation studies on the IRES target have been performed using antisense, aptamer, ribozyme and siRNA approaches. At least one peptide and three classes of small-molecules have been described as inhibitors of in vitro IRES activity, including biaryl guanidines, phenazine derivatives, and vitamin B12.
The 3′ NTR is comprised of three distinct domains including a 40-nt variable region, a downstream poly(U/C) tract of heterogeneous length, and a highly conserved 98-nt segment termed X-region. Both the poly(U/C) tract and the X-region are essential for RNA replication but not for translation. Secondary structure prediction, phylogenetic analyses, as well as nuclease probing suggest that the X-region folds into three stem-loops which are the most conserved RNA sequences in the HCV 3′-NTR. It has been suggested that specifically stem-loop 1 is involved in replication by providing binding sites for the viral NS3 protease/helicase and NS5B polymerase. Cellular factors, including polypyrimidine tract-binding protein (PTB) and ribosomal proteins, have been shown to interact with the X-region RNA, thereby interfering with the binding of viral proteins and participating in the regulation of viral translation. The stem-loops 2 and 3 were mapped as essential parts of the PTB binding site. The highly conserved secondary structure of the X-region as well as its importance for RNA replication and as binding site for viral and host proteins have led to suggestions to exploit the 3′ NTR as a target for antiviral agents including antisense oligonucleotides.
Evidence has emerged that stem-loop 2 in the X-region might participate in a pseudoknot interaction with a conserved RNA element within the coding region of the viral NS5B polymerase. Earlier phylogenetic and RNA folding analyses suggested the presence of several stem-loop structures within the NS5B coding region. Four of the predicted stem-loops that are located within a highly conserved region of the HCV genome were confirmed by mutational and biochemical analyses. The secondary structure of stem-loop V (5BSL3.2) was also confirmed by NMR spectroscopy. The stem-loops V and VI are essential for viral RNA replication and thus constitute cis-acting replication elements (CRE) which are similar to cis-acting RNA structures found in the genomes of other RNA viruses. The HCV NS5B polymerase, which has been shown to interact with 3′ viral genomic RNA, binds specifically to SL-V. While the precise function of the SL-V RNA element has yet to be determined, the role it plays in viral replication is dependent on its location within the HCV genome. This context dependence of SL-V function is likely to be related to a kissing interaction between the apical hairpin loops of SL-V and SL-2 in the HCV X-region which gives rise to a pseudoknot structure involving coding region and 3′ NTR of the viral genome. It has been speculated that formation of a replication-essential pseudoknot might include interactions with NS5B polymerase. Despite the current lack of extensive functional insight into the role of conserved RNA elements in the NS5B coding region, the essentiality of these CRE for viral replication renders them promising targets for RNA-directed antiviral drugs.